Transfer of information between two neurons or order cross from a neuron to a target cell, such as a motor neuron and a contractil muscular fiber, occurs through junctions named synapsis.
The main synaptic way of communication implies emission of chemical molecules or neurotransmitters from nerve terminals of the transmitter neuron (presynaptic neuron), which are taken up by the receiving postsynaptic cell. Acetylcholine intervenes as a neurotransmitter between motor neurons and streaked skeletal muscles for example.
In nerve terminals, chemical molecules are stocked into synaptic vesicles which are made of small and spherical organelles of 50 nm in diameter, delimited by a lipidic membrane. Those molecules are up to 500 000 in nerve terminals. To command a volountary impulse or movement or to keep a position, contraction order is given to muscles by way of several bioelectrical impulsions. Each impulsion spreads up to nerve terminals of motoneurons and depolarizes them. Synaptosomal associated proteins, namely ionic chanels, are activated and rendered permeable to calcium ions which get in the nerve terminals. This induces the fusion of synaptic vesicles with nerve terminal membranes. By this way, vesicles neurotransmitters content is released through the synaptic slot between transmitter neuron and target muscular fiber. This release is called exocytose. Acetylcholine diffuses through the synaptic slot and then molecules are detected by acetylcholine receptors localized on muscular fibers. This detection induces a postsynaptic signal, namely a depolarization, which creates an action potential.
Sometimes this mechanism of signal transduction is deficient. Amyotrophic lateral sclerosis is a devastating neurodegenerative disorder, affecting the motor neurons of the central nervous system (cortex, brainstem) and the peripheral motor neurons (spinal cord). The disease destroys the nerve cells that control voluntary movement and is characterized by progressive muscle weakening, paralysis and death, usually within 2 to 5 years after the appearance of the first clinical sign. The disease affects limbs, tongue, pharynx and larynx muscles. Onset usually occurs after age 45, and the rate and pattern of disease progression vary widely.
There is no treatment for this disease and its etiology remains unknown, although the discovery of missense mutations in the gene for copper-zinc superoxide dismutase (SOD1) in some pedigrees with familial ALS (FALS) has marked an important advance in the understanding of ALS physiopathology. Most ALS cases are sporadic (SALS), and of the 10% autosomal dominant inherited cases, about 20% of kindreds are associated with mutations in the SOD1 gene. Over 60 point mutations have been identified to date in all five exons of the SOD1 gene, involving 43 of the 153 residues.
The SOD1 enzyme plays a critical role in preventing cell damage by free radicals, by scavenging the superoxide anion radical, converting it into oxygen and hydrogen peroxide. Although the mechanism by which mutations in the gene encoding ubiquitous SOD1 protein lead to selective motor neuron degeneration is unknown, some such mutations cause motor neuron disease when expressed in transgenic mice. For example, G93A mutant mice (glycine to alanine substitution at position 93) develop progressive loss of motor neurons and vacuolar degeneration of mitochondria within motor neuron cell bodies of the spinal cord and the brainstem leading to a progressive decline in motor function and death at 5 to 6 months of age. Novel cytotoxic properties of the mutated SOD1, rather than a decrease in enzyme activity, are thought to be involved in this neurotoxicity. In particular, the SOD1 mutation may induce misaccumulation of the neurofilaments (NF), as has been described in both human and experimental ALS and a lower level of motor neuron degeneration combined with delayed progression of the disease has been reported in mice carrying both a SOD-1 mutation and a disrupted NF-L gene.
Others diseases such as spinal muscular atrophy (SMA), epilepsy, Parkinson's disease or Alzheimer's disease are also caused by neurological disorders. Furthermore, trauma associated with the spinal cord can induce neurological disorders.
A number of neurotrophic, neuroprotective and growth factors are potential candidates for treating ALS and other Motor Neuron Diseases (MND) (Alisky and Davidson, 2000). However, these factors delivered systemically have not been beneficial to patients in clinical trials. The reasons for this lack of success include limited access to motoneurons, insufficient specificity, or down-regulation of binding sites (Sendtner, 1997). Therapeutic gene transfer offers potential advantages over direct administration of the protein, such as continuous and/or targeted production of the desired transgene in vivo. The continuous in situ production of physiological concentrations of growth factors by gene transfer may allow the expression of the potential therapeutic effect of such molecules (Gravel et al., 1997; Alisky and Davidson, 2000). Retrograde axonal transport of recombinant adenoviral vectors has been used successfully to deliver genes to motoneurons in mammalians following injection of the vectors into muscles (cf: WO 98/31395). However, it seems that only a small proportion of motoneurons take up and retrogradely transport adenoviral particles.
Intramuscular injection of recombinant adenoviruses is an approach particularly well-suited to gene therapy of MND because it allows motoneuronal transduction and production of the therapeutic proteins in the Central Nervous System (CNS) after axonal retrograde transport of the vectors (Finiels et al., 1995; Ghadge et al., 1995). However, several recent studies reported that only a low percentage of motoneurons were transduced following peripheral administration of recombinant adenoviruses (Gravel et al., 1997; Perrelet et al., 2000). Intramuscular injection of recombinant adenoviruses into the facial musculature or into the tongue of mice resulted in less than 10% of motoneurons being transduced (Gravel et al., 1997). This could be due to only a subpopulation of spinal motoneurons being susceptible to infection (Perrelet et al., 2000). In agreement with these studies, a low rate of motoneuron transduction (4%) has also been observed in the mouse hypoglossal nucleus after injection of the Ad-RSV-βgal adenovirus into the tongue.
Direct intracerebral injection of adenoviral vectors into various brain structures allows the transfer of a therapeutic gene both at the injection site and also at distance, via neurons that send axonal projections to the injection site (Akli et al., 1993; Davidson et al., 1993; Le Gal La Salle et al., 1993). This observation suggests that adenoviral particles are taken up at nerve terminals and are retrogradely transported to the neuronal cell bodies. For example, neurons located in the substantia nigra or in the inferior olive can be efficiently transduced by inoculation of the striatum and the cerebellum, respectively, with the vectors (Akli et al., 1993; Ridoux et al., 1994). This remarkable property renders recombinant adenoviruses particularly useful for retrograde neuronal tracing in the CNS (Ridoux et al., 1994; Kuo et al., 1995). Another application of this property is for the transduction of the not easily accessible motoneurons by peripheral injection of the vectors (Finiels et al., 1995; Ghadge et al., 1995). This route of administration is particularly suitable for treating fatal neurodegenerative diseases affecting motoneurons (Alisky and Davidson, 2000). It is a preferable alternative to more invasive intramedullar injections with gene vectors. However, as indicated above, the percentage of motoneurons transduced is low, even when large doses of the vector are used (Gravel et al., 1997; Perrelet et al., 2000). Another potential problem is ectopic production of the exogenous protein, i.e., the presence of the protein in the muscle may result in side effects.
The invention now provides an improvement to gene delivery to motor neurons, particularly to the “retrograde transport approach” and allows an overexpression of any polypeptide or nucleic acid in said neurons in vivo. The invention stems from the use of various compounds that cause synaptic nerve sprouting, which significantly improve retrograde transport and gene expression into neurons. The invention also discloses improved vectors designed to specifically transduce the neurons when injected into the brain or muscles which further improve the efficacy, selectivity and safety of the proposed methods.